The genome sequence of the Fig-leaf Skeletoniser, Choreutis nemorana (Hübner, [1799])

We present a genome assembly from an individual male Choreutis nemorana (the Fig-leaf Skeletoniser; Arthropoda; Insecta; Lepidoptera; Choreutidae). The genome sequence is 300.2 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.52 kilobases in length. Gene annotation of this assembly on Ensembl identified 15,351 protein coding genes.


Background
Choreutis nemorana (Hübner, [1799]) also known as the Fig-leaf Skeletoniser (Sterling et al., 2023), is a moth in family Choreutidae with a wingspan of 16-20 mm (8-10 mm forewing length; Stojanović et al., 2020;Sterling et al., 2023).Its forewing has a rounded costa, with apex slightly projecting and sinuate termen accentuated by its white fringe, and its background colour is usually orange-brown (or sometimes dark greyish) with four irregular thin white crosslines (the last submarginal) and the subterminal area is usually darker orange.Like other choreutids, it holds its wings upwards and somewhat furled when active and runs in a jerky motion on the leaf surface.The legs are clothed in white scales as if covered in fungus, perhaps advantageous for winter hibernation.C. nemorana differs from other Choreutidae (at least among European species) in its orange wing colouration which distinguishes it from the otherwise similar C. pariana (Clerck, 1759).The species is particularly prevalent in the Mediterranean area where its foodplant naturally occurs, also occurring in northwestern Africa (De Prins et al., 2014).Elsewhere, the species is widespread from the Macaronesian islands, through Europe to western Asia including Asia Minor, Caucasus, Turkmenistan and Uzbekistan (west of the Caspian Sea) but its distribution does not extend as far north as Scandinavia (GBIF Secretariat, 2024); however, there are two records from China (De Prins et al., 2014).There are strong signs that the species is spreading rather rapidly north and west (Stojanović et al., 2020), being found first in Belgium in 2009 (De Prins et al., 2014); Lepiforum (2024) gives a summary by country of its current distribution in Europe.
C. nemorana feeds exclusively as a larva on Ficus (Moraceae) and is apparently monophagous on F. carica L., being considered a minor pest of figs (Alford, 2007); it can occasionally cause extensive damage to F. carica plants in the London area and in Europe (Ellis, 2024).Its spread may have been exacerbated by a combination of the plant trade (De Prins et al., 2014) and global warming (Stojanović et al., 2020).
The greenish yellow larva, with each thoracic and abdominal segments bearing about eight black shiny pinacula, has been found in the UK from late May to early November, feeding underneath a web on the upper surface of leaves of Ficus carica where its eggs are laid (De Prins et al., 2014) and which the larvae skeletonise (Sterling et al., 2023).The larvae are very active if disturbed and may drop to the ground (De Prins et al., 2014).The brownish black pupa is found in a whitish silken fold at the leaf margin.The diurnal adult which appears in at least two broods between July to October (Norfolk Moths, 2024); the species is bivoltine in Europe (De Prins et al., 2014).The moth hibernates as an adult, including in thatch and among dead leaves in hedgerows (De Prins et al., 2014).The adult nectars on a wide range of flowers.

Genome sequence report
The genome was sequenced from a male Choreutis nemorana (Figure 1) collected from Kensington Gardens, London, UK (51.51, -0.17).A total of 77-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 6 missing joins or mis-joins, reducing the scaffold number by 1.03%, and increasing the scaffold N50 by 0.23%.
The final assembly has a total length of 300.2 Mb in 95 sequence scaffolds with a scaffold N50 of 10.2 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.25%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome.Chromosomescale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
A Choreutis nemorana caterpillar (specimen ID NHMUK013696811, ToLID ilChoNemo1) was collected by hand from Kensington Gardens, London, UK (latitude 51.51, longitude -0.17  Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific

Software tool Version
Operations core at the WSI on a Pacific Biosciences SEQUEL II instrument.Hi-C data were also generated from remaining tissue of ilChoNemo1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant software tool versions and sources.

University of Cologne, Cologne, Germany
This genome report describes and summarizes the details of a de novo genome assembly of the moth species Choreutis nemorana.The value and relevance of novel de novo assembly should not be questioned in general and this particular genomes will contribute to future research on insect/lepidopteran phylogenies and functional studies.
I highly recommend to include a nicer and more informative picture that shows the adult moth.Especially, since it has been described in its adult morphology in the background section.The provided photo is a visual voucher of the sequenced individual but not really representative for the species.
The assembly pipeline applies state-of-the-art approaches and the results are documented according to standard.It can be recommended to use Braker3 for future annotations, as here the pipeline used Barker2.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: biodiversity genomics, ecological genomics, population genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
In the Genome sequence report the first sentence can be written as "The genome was sequenced from a male caterpillar of C. nemorana…….."

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In the Methods the genus and species names were merged.The genus name and species can be separated.The sentence can be written as "A C. nemorana caterpillar (specimen ID NHMUK013696811, ToLID ilChoNemo1) was collected from Kensington….
○ Above all, I confirm that the manuscript meets the necessary scientific standard and is suitable for Indexed".

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes

C
. nemorana is a native of Europe, first adventive to the UK in 2014 (Kensington, Gardens, London) (De Prins & De Prins, 2014) and is now spreading rapidly, recorded in Kent, Hertfordshire, East Anglia, Hampshire, Dorset and the Isle of Wight.The first reported breeding population in the UK was found in Kensington Gardens, London (De Prins & De Prins, 2014).
C. nemorana is a species that is somewhat isolated from other Choreutidae, falling over 7.1% pairwise divergent in COI-5P to C. pariana and C. aegyptiaca (Zeller, 1867).In the phylogenetic analysis of Rota et al. (2016), C. nemorana is recovered as sister to a distal clade among Choreutis that includes the Oriental C. euclista (Meyrick, 1918), the Palaearctic C. aegyptiaca, C. pariana, C. diana (Hübner, 1822) and eight other species, with an estimated divergence of around 17-20 Ma.The mitogenome from the genome assembly (OX438626.1)represents for COI-5P the most common haplotype from Europe on BOLD (28/03/2024) and belongs to the single BIN cluster BOLD:AAL6537.The nearest neighbour on BOLD is Choreutis sp.'JDP1' of Rota et al., 2016 (KT956504, BOLD: ADR7873).The genome will be helpful, e.g. in further molecular taxonomic work on Choreutis.

Figure 2 .
Figure 2. Genome assembly of Choreutis nemorana, ilChoNemo1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 300,258,981 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (16,217,284 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (10,167,060 and 6,945,856 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CASGFM01/dataset/CASGFM01/snail.
) on 2021-07-13.The specimen was collected and identified by David Lees (Natural History Museum) and preserved by dry-freezing at -80 °C.The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.The sample was prepared for DNA extraction at the WSI Tree of Life Core Laboratory.The ilChoNemo1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the

Figure 3 .
Figure 3. Genome assembly of Choreutis nemorana, ilChoNemo1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CASGFM01/dataset/CASGFM01/blob.

Figure 4 .
Figure 4. Genome assembly of Choreutis nemorana, ilChoNemo1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CASGFM01/dataset/CASGFM01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Choreutis nemorana, ilChoNemo1.1:Hi-C contact map of the ilChoNemo1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=d4lxwUO7RY--rBe8qZtHdQ.

Darwin Tree of Life Project Sampling Code of Practice', which
was used in the default protein mode to generate annotation for the Choreutis nemorana assembly (GCA_949316135.1) in Ensembl Rapid Release at the EBI.can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.